In the human body two hormones, parathormone and calcitonin, along with vitamin D have major roles in controlling the metabolism of calcium (Ca.sup.++) and phosphate (Pi). Binding of parathormone to kidney cells yields among other things increased renal tubular reabsorption of Ca.sup.++ and Mg.sup.++, and enhanced excretion of Pi. The rates of renal gluconeogenesis and renal respiration are increased and hydroxylation of vitamin D to its active form (1,25-dihydroxyvitamin D) is increased. In bones, parathormone inhibits collagen synthesis in active osteoblasts, increases osteocytic and osteoclastic osteolysis and increases the rate of maturation of osteoblasts and osteoclasts. There is increased mobilization of Ca.sup.++ from bone and increased transport of Ca.sup.++ across the intestine, accompanied by elevation of plasma Ca.sup.++. Calcitonin reduces bone resorption, perhaps by inhibiting osteocytes and osteoclasts. This is accompanied by hypocalcemia and hypophosphatemia. Enhanced excretion of Pi may be a secondary response to alterations in plasma Ca.sup.++. Secretion of parathormone is reduced and that of calcitonin is increased in response to elevated Ca.sup.++ in the circulation.
In the presence of calcitonin, Ca.sup.++ and Pi are deposited in bone. The exact identities of the intermediates involved in the early steps of mineral deposition are still not known with certainty. It appears that brushite, CaHPO.sub.4.sup.. 2H.sub.2 O, is deposited first but that it redissolves and is converted to amorphous calcium phosphate, a noncrystalline association of ions. This is believed subsequently to yield octacalcium phosphate, Ca.sub.8 (HPO.sub.4).sub.2 (PO.sub.4).sub.4.sup.. 5H.sub.2 O, which in turn is converted to hydroxyapatite, Ca.sub.10 (OH).sub.2 (PO.sub.4).sub.6, the least soluble mineral of the group.
Several pathological conditions in which bone mineralization is deficient have been observed. These can arise from a number of conditions including, for example, abnormalities in hormonal regulation, renal cortical damage and dietary Ca.sup.++ and phosphate deprivation. The example of osteoporosis is given for illustration.
Osteoporosis is characterized by reduced mineral content of bone, especially trabecular bone, and is particularly prevalent in postmenopausal women. The underlying causes of osteoporosis in the female appear first to arise at about the age of 30-40 years with reduced secretion of calcitonin. This is accompanied by hypocalcemia and subsequent elevation of parathormone. The 1 alpha-hydroxylase activity of the kidney becomes elevated with a probable resulting increase in "remodeling" of mineral deposits, especially in trabecular bone. The skeletal complications of osteoporosis become apparent after the secretion of estrogen begins to decrease (i.e. 40-50 years of age). The metabolism of vitamin D appears to be reduced when the estrogen levels are lower and the intestinal absorption of calcium is affected. The reduced intestinal absorption of calcium appears to place an increased demand on the skeletal system, which in time yields reduced mineral deposits in trabecular bone. This results, for example, in an increased incidence of fractures of the hip and vertabrae. Similar demineralization also can arise from hyperparathyroidism and chronic renal insufficiency.
There is at the present time a clinical need for a rapid assay of the mineral content of bone. The physical and chemical properties of mineral deposits in bone severely limit the physical techniques by which such deposits can be detected. All prior art techniques for bone analysis have serious limitations. Ashing and extraction normally are prerequisites to elemental analysis, and these can result in incomplete recovery of individual components. Furthermore, total phosphate analyses are seriously affected by procedures used routinely in the clinical setting to treat patients. Thus the results of phosphate analyses do not necessarily yield a reliable indication of variations in bone mineralization.
Clinical X-ray procedures provide only a measure of "bone density" toward transmittance of this radiation, which is not indicative of mineral composition. Procedures such as scanning electron microscopy and X-ray crystallography have been used experimentally, but these techniques are cumbersome and time-consuming to perform. Furthermore, the results from these two techniques have not been in full agreement.
In may earlier application an assay is disclosed which includes .sup.31 P nuclear magnetic resonance (NMR) spectroscopy of bone biopsy samples utilizing cross polarization, magic angle sample spinning and/or dipolar proton decoupling techniques to provide reliable characterization of mineral composition of excised biological samples. The NMR assay is rapid, requires a minimum of sample preparation, and provides the chemical composition of the mineral deposit.
All of the above assays with the exception of clinical radiology, however, require that a bone biopsy be removed from the patient. The taking of such a bone biopsy is very painful and distressing to the patient, and there is a high likelihood that an infection will develop at the locus of tissue extraction. A more desirable approach would be to perform a noninvasive assay of the bone while it is still in the patient. This is especially true if routine screening of women is done to detect osteoporosis early in its onset when it can be treated effectively.